Project Summary Our goal is to develop a novel L. lactis probiotic-based therapeutic for the treatment of Sjgren's Syndrome (SjS). SjS is a progressive, chronic autoimmune disease characterized by inflammatory cell infiltration of the salivary and lacrimal glands, resulting in acinar epithelial cell atrophy, cell death, and loss of exocrine function1- 6. It is a debilitating disease affecting as many as 3.1 million individuals in the US7-8, with women being nine times more likely to be afflicted with SjS than men5, 8-9. Treatment of SjS remains a significant unmet medical need. Current treatment relies on replacement therapies such as artificial saliva and eye lubricants or immunosuppressive agents10-11. Because of the multiple antigens involved in this disease process, i.e., ?-fodrin12-15, ribonuclear protein Ro/SSA12, 16-18, La/SSB 12, 16-17, and M3R17, 19-20, oral tolerance methods become problematic. Thus, the capacity to stimulate regulatory cells independent of knowing the antigen specificity for the disease poses as an attractive therapeutic device. Originally conceived as a diarrheal vaccine for humans21-23, we found colonization factor antigen I (CFA/I) from human enterotoxigenic E. coli (ETEC), is potently effective in preventing and treating experimental models for multiple sclerosis24-26 and arthritis27-29. In fact, purified CFA/I fimbriae, when given orally or nasally, and administered in lieu of live Salmonella-CFA/I as a source of fimbriae, can effectively attenuate inflammation without the associated side-effects from Salmonella27. To avoid efforts and costs associated with producing sufficient quantities of recombinant fimbriae, we have successfully engineered a Lactococcus lactis strain to express CFA/I fimbriae (L. lactis-CFA/I). Retaining its inhibitory activity, L. lactis-CFA/I can protect against collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). More importantly, our preliminary data now shows L. lactis-CFA/I reverses genetically induced SjS. Based on these findings, our goal is to develop L. lactis-CFA/I as an oral therapeutic to arrest SjS.! This application is focused on measuring the efficacy, PK/PD and safety of VTC-CFA to support further product development. The specific aims are to: 1) produce sufficient amounts of VTC-CFA and establish analytical and bioactivity assays; 2) determine the pharmacokinetics (PK) and optimal oral dosing of VTC-CFA in mice, and 3) establish the acute toxicology and MTD profiles for VTC-CFA in mice. Successful commercialization of VTC- CFA would ultimately provide a profound front-line medical advancement in the treatment of SjS.